EP4278227A1 - Dispositif de microscope - Google Patents

Dispositif de microscope

Info

Publication number
EP4278227A1
EP4278227A1 EP22700481.9A EP22700481A EP4278227A1 EP 4278227 A1 EP4278227 A1 EP 4278227A1 EP 22700481 A EP22700481 A EP 22700481A EP 4278227 A1 EP4278227 A1 EP 4278227A1
Authority
EP
European Patent Office
Prior art keywords
light
microscope device
sample
dichroic
reflection element
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22700481.9A
Other languages
German (de)
English (en)
Inventor
Rainer PROF UHL
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Miltenyi Biotec GmbH
Original Assignee
Miltenyi Biotec GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Miltenyi Biotec GmbH filed Critical Miltenyi Biotec GmbH
Publication of EP4278227A1 publication Critical patent/EP4278227A1/fr
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G02B21/361—Optical details, e.g. image relay to the camera or image sensor
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/10—Beam splitting or combining systems
    • G02B27/1006—Beam splitting or combining systems for splitting or combining different wavelengths
    • G02B27/1013—Beam splitting or combining systems for splitting or combining different wavelengths for colour or multispectral image sensors, e.g. splitting an image into monochromatic image components on respective sensors
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/10—Beam splitting or combining systems
    • G02B27/14—Beam splitting or combining systems operating by reflection only
    • G02B27/141—Beam splitting or combining systems operating by reflection only using dichroic mirrors
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B2207/00—Coding scheme for general features or characteristics of optical elements and systems of subclass G02B, but not including elements and systems which would be classified in G02B6/00 and subgroups
    • G02B2207/113—Fluorescence

Definitions

  • the invention relates to a camera-based microscope which employs four-color distinction capabilities to provide a maximally contrast-rich fluorescence respectively transmitted light image.
  • a multicolor microscope is disclosed in WO 2020/038752, which relates to a microscope device having dual emission detection capabilities. It employs two cameras and places a dichroic beam splitter into the finite optical space between the microscope and the two cameras, which record the two desired spectral regions. In order not to distort the transmitted spectral image, the dichroic is kept as thin and the reflection angle as small as possible. However, since a thin substrate tends to compromise flatness and hence the quality of the reflected image, an optimal thickness always reflects a compromise between the quality of the transmitted and the reflected image.
  • Object of the invention is therefore a microscope device comprising a microscope objective (1); one or more light sources; at least 3 dichroic beam splitters (50, 51, 56) and at least 2 cameras (109, 117) characterized in that the light generated by the light source interacts with the sample (3) thereby producing a sample beam (6), wherein
  • - sample beam (6) is divided with a first dichroic beam splitter (50) into beam (K) and beam (L) wherein beam (K) and beam (L) have different spectral ranges of light and wherein
  • - beam (K) is divided with a second dichroic beam splitter (51) into a first beam (A) having a first spectral range of light and a second beam (B) having a second spectral range of light and wherein first beam (A) is guided via reflection element (54) on the detector of the first camera (109) and wherein second beam (B) is guided via reflection elements (52) and (53) on the detector of the first camera (109) and wherein
  • - beam (L) is divided with a third dichroic beam splitter (56) into a third beam (C) having a third spectral range of light and a fourth beam (D) having a fourth spectral range of light and wherein third beam (C) is guided via reflection element (58) and (59) on the detector of the second camera (117) and wherein the fourth beam (D) is guided via reflection element (57) on the detector of the second camera (117).
  • Such microscope devices are especially useful for detecting multiple spectral ranges emitted by a sample which is often the case in sequencing DNA/RNA molecules. To avoid damaging of the sample, the interaction between the light and the sample should be kept as short as possible. Since the device of the invention can detect at least 4 different spectral ranges simultaneously, the use of the microscope device as disclosed herein in a sequenc- ing-by-synthesis process it is a further object of the invention.
  • FIG. 1 and 2 show two embodiments of the path of light of the microscope of the invention
  • FIG. 3 shows a part of the device upstream of sample beam (6)
  • Fig. 1 or 2 In the device of the invention as shown in Fig. 1 or 2, light originating from the sample (either transmitted or emitted) is separated into four spectral regions by three dichroic beam splitters, and the resulting image beams A, B, C and D are directed to two cameras (109, 117), whereby each of the two cameras records two spectral regions side-by-side on its sensor-chip.
  • high-end camera chips are available for image fields much bigger (for example 36 x 24 mm) than the image field of current microscopes, which maximally covers a square field of 17 x 17 mm.
  • TDI cameras are used.
  • the microscope according to the invention may comprise a at least one camera (109, 117) which is of charge-coupled device (CCD) type, electron-multiplying charge-coupled device (EM-CCD) type, complementary metal-oxide-semiconductor (CMOS) type, scientific complementary metal-oxide-semiconductor (CMOS) type, time delayed integration (TDI) type or a combination thereof.
  • CCD charge-coupled device
  • E-CCD electron-multiplying charge-coupled device
  • CMOS complementary metal-oxide-semiconductor
  • CMOS scientific complementary metal-oxide-semiconductor
  • TDI time delayed integration
  • the microscope is equipped to detect four spectral ranges, for example 405, 488, 561 and 638 nm or 375 nm, 473 nm, 532 nm, und 660 nm.
  • the cut-on wavelengths are chosen to 488, 561 and 638 nm as to separate the emission into the four spectral regions between the excitation wavelengths.
  • the respective spectral ranges of light of the first, second, third and fourth beams are between 473 nm and 532 nm; 532 nm and 594 nm, 594 nm and 660 nm; 633 nm and 660 nm. It should be noted that these spectral ranges of light are given by way of example and depend on the function of the beam splitters, i.e. are not necessary bounded to this sequence.
  • At least one of the dichroic beam splitters (50, 51, 56) is arranged (tiled) in the path of light as to minimize or avoid the chromatic error of the light detected by the camaras. Since the chromatic error depends on the spectral range / the wavelength of the light, the angular position of the dichroic beam splitters (50, 51, 56) may be same or different.
  • the tilt angles of first, second and third dichroic beam splitter (50, 51, 56) with the respective residual images may be independently between + 45° and - 45°, preferable independently between + 30° and - 30° or independently between + 25° and - 25°. or independently between + 15° and - 15°.
  • the tilt angles of the first and third dichroic beam splitters (50, 56) with the respective residual beams may be in opposing directions.
  • the light sources preferable provide light having a spectral range of wavelengths of 300 to 1750 nm, preferable 300 - 800 nm like white light, laser light, or LED light.
  • the sample may be subjected to the light “as is” or may be provided with fluorescence or phosphorenscence agent to mark regions or interest. To avoid damaging the sample, preferable light sources producing light with longer wavelengths are used, such as 525 and 635 nm.
  • the sample beam may be or comprise fluorescence or phos- phorenscence radiation originating from the sample (3) or radiation transmitted or reflected by the sample (3).
  • the microscope device may be provided with at least one focusing element (2) into the beam-path of the sample beam upstream of the first beam splitter (shown in Fig. 1).
  • two or more focusing elements (2) may be provided in the sample beam downstream of the first beam splitter as shown in Fig. 2.
  • the focusing elements may consist or comprise at one lens or at least one objective or a combination thereof.
  • the microscope device according to the invention may be provided with one or more optical element (21, 22, 23, 24) into the beam-path of first, second, third and/or fourth image beams A, B, C, D.
  • optical elements are capable of focal plane or image plane shift and can optionally be inserted and removed from the beam-paths with an appropriate device.
  • Suitable optical elements have a higher refractive index than the surrounding medium and may consist of coated or uncoated glass or polymer.
  • the optical elements can be provided with a filter for chromatic correction of any optical distortion which caused by the dichromatic beam splitters
  • Fig. 1 and 2 show the device of the invention, which uses two identical optical arrangements (48) and (49) for each of the two camera-chips (109) and (117).
  • beam splitter (51) splits K into beams A and B
  • a beam splitter (56) splits L into beams C and D.
  • the reflected beam (B) needs two more reflections at mirrors (52) and (53), whereas the transmitted beam (A) needs only one reflection at surface (54), before it reaches the detector (109).
  • the beam transmitted by dichroic (50) is split into (C) by being reflected at dichroic (56), and into (D), which is transmitted at dichroic (56).
  • the transmitted fraction (D) requires a single reflection at a reflecting element (57), whereas the reflected fraction (C) requires two more reflections at reflecting surfaces (58) and (59).
  • all beams reflected by a dichroic beam splitter may carry ghost-image information.
  • the tilt-angle of the two dichroic elements (50) and (51) is kept at about 25° and at opposing angles, as to compensate for chromatic aberrations.
  • the thickness of the dichroic beam splitters is kept as small as possible (usually 1-3 mm) as to minimize thickness-related aberrations in transmission, but thick enough to maintain flatness of the reflecting surfaces, which is of paramount importance for image quality of the reflected fraction of the beam. (Fig. 1).
  • one dichroic element is positioned between the objective lens (1) and the focusing element (2), allowing for larger tilt angles (preferably 45°) at this dichroic position and allowing for larger thickness (usually 3 mm, possibly 1 mm to 5 mm).
  • the cure for this is to bring appropriate bandpass filters or optical elements (21, 22, 23 and 24) into the beam-path.
  • the reflection element (52) and/or (53) may be provided with a filter layer having the same optical properties as first dichroic beam splitter (50) and/or as second dichroic beam splitter (51).
  • the reflection element (54) may be provided with a filter layer having the same optical properties as first dichroic beam splitter (50).
  • the reflection element (58) and/or (59) may be provided with a filter layer having the same optical properties as third dichroic beam splitter (56).
  • reflection element (52) and/or reflection element (53) may be provided with a filter layer having the same optical properties as second dichroic beam splitter (51).
  • the microscope device of the invention may be provided with at least one focusing element (2) into the beam-path of the sample beam in order to create an im- age (6).
  • the least one focusing element (2) may be provided into the beam-path of the image L and/or K.
  • the focusing element (2) may consist or comprise at one lens or at least one objective or a combination thereof.
  • a short-pass filter can replace the bandpass, if the dichroic beam splitter is a short-pass, one needs long- pass filter.
  • the microscope device allows to differentiate the image of a color-labelled object with respect to up to four spectral regions, both in fluorescence-emission and in transmitted light absorption.
  • the optical path-lengths are identical for all spectral ranges (color-channels) and all images lie in the plane of the respective detector chip (camera).
  • the optical layout may be used to correct for longitudinal color-imperfections by adjusting the optical path-lengths accordingly.
  • a volitional detuning of the path-lengths may be used to look at two or more focal depths simultaneously and to reconstruct contrast-enriched images from images taken at different focal positions.
  • a dichroic ensemble designed for separating the emission excited by a 405 and a 488 nm laser, divides the light of a white light emitting diode into two spectral regions below and above 488 nm.
  • the thickness of the optical element (22) determines the path-length difference of beams A and B.
  • optical element (21), (23) and/or (24) can be provided with optical element (21), (23) and/or (24).
  • the microscope devices of the invention are especially useful in methods for detecting multiple spectral ranges which are emitted during sequencing of DNA/RNA molecules, in particular for sequencing -by- synthesis processes to obtain DNA or RNA sequence information of a biological sample.
  • the sequencing-by- synthesis process is performed by hybridization of nucleotides provided with different dyes to the DNA or RNA of the biological sample and wherein the dyes emit light upon excitation by the one or more light sources in the spectral ranges A, B, C and D or combinations thereof.
  • the dyes emit light upon excitation by the one or more light sources in the spectral ranges A, B, C and D or combinations thereof.

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  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Multimedia (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Microscoopes, Condenser (AREA)

Abstract

L'invention concerne un dispositif de microscope comprenant un objectif de microscope (1) ; une ou plusieurs sources de lumière ; au moins trois séparateurs de faisceau dichroïque (50, 51, 56) et au moins deux caméras (109, 117) caractérisées en ce que la lumière générée par la source de lumière interagit avec l'échantillon (3) ce qui permet de produire un faisceau d'échantillon (6), le faisceau d'échantillon (6) étant divisé par un premier diviseur de faisceau dichroïque (50) en faisceau (K) et le faisceau (L), le faisceau (K) et le faisceau (L) ayant différentes plages spectrales de lumière, et le faisceau (K) étant divisé par un deuxième diviseur de faisceau dichroïque (51) en un premier faisceau (A) ayant une première plage spectrale de lumière et un deuxième faisceau (B) ayant une deuxième plage spectrale de lumière et le premier faisceau (A) étant guidé par l'intermédiaire d'un élément de réflexion (54) sur le détecteur de la première caméra (109) et le deuxième faisceau (B) étant guidé par l'intermédiaire d'éléments de réflexion (52) et (53) sur le détecteur de la première caméra (109), et le faisceau (L) étant divisé par un troisième séparateur de faisceau dichroïque (56) en un troisième faisceau (C) ayant une troisième plage spectrale de lumière et un quatrième faisceau (D) ayant une quatrième plage spectrale de lumière et le troisième faisceau (C) étant guidé par l'intermédiaire d'un élément de réflexion (58) et (59) sur le détecteur de la deuxième caméra (117) et le quatrième faisceau (D) étant guidé par l'intermédiaire d'un élément de réflexion (57) sur le détecteur de la deuxième caméra (117). L'invention concerne également l'utilisation du microscope pour obtenir des informations de séquençage.
EP22700481.9A 2021-01-12 2022-01-12 Dispositif de microscope Pending EP4278227A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102021100350 2021-01-12
PCT/EP2022/050491 WO2022152720A1 (fr) 2021-01-12 2022-01-12 Dispositif de microscope

Publications (1)

Publication Number Publication Date
EP4278227A1 true EP4278227A1 (fr) 2023-11-22

Family

ID=79927066

Family Applications (1)

Application Number Title Priority Date Filing Date
EP22700481.9A Pending EP4278227A1 (fr) 2021-01-12 2022-01-12 Dispositif de microscope

Country Status (5)

Country Link
US (1) US20240068026A1 (fr)
EP (1) EP4278227A1 (fr)
JP (1) JP2024502383A (fr)
CN (1) CN116830008A (fr)
WO (1) WO2022152720A1 (fr)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009544988A (ja) * 2006-07-20 2009-12-17 コーニンクレッカ フィリップス エレクトロニクス エヌ ヴィ マルチカラーバイオセンサ
EP3243106A4 (fr) 2015-03-23 2018-10-03 East Carolina University Systèmes de séparation de faisceau à longueurs d'ondes multiples pour l'imagerie simultanée d'un objet distant dans deux canaux spectraux ou plus à l'aide d'une seule caméra
EP3614191A1 (fr) 2018-08-20 2020-02-26 Till GmbH Dispositif de microscope

Also Published As

Publication number Publication date
US20240068026A1 (en) 2024-02-29
JP2024502383A (ja) 2024-01-18
WO2022152720A1 (fr) 2022-07-21
CN116830008A (zh) 2023-09-29

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